The transmission of biologically active materials to cells is an essential component of a wide range of therapies. Such therapies include supplying a cell with a protein having a necessary biological activity, providing a nucleic acid (i.e., DNA, RNA, cDNA) molecule to a cell (gene therapy), immunizing a subject against a foreign protein (vaccination), immunizing a subject against a foreign protein by introducing a gene that encodes for the protein (gene vaccination), and inhibiting the production of a protein in a cell by providing the cell with a nucleic acid molecule that is antisense i.e., complimentary, to mRNA encoding the protein or otherwise interfering with the mRNA encoding the protein.
There are, however, several obstacles to delivery of such agents to a cell, including the fact that the phospholipid bilayer containing the outer membrane of most cells prohibits indiscriminate entry of materials into the cell. Approaches described to introduce active agents into cells include, for example, microinjection and electroporation. Other approaches involve viral vectors and chemical-mediated introduction.
Another approach for delivery of active agents to cells, described in the art, is liposome-based delivery. In particular, delivery of genetic material to cells using liposomes has been widely studied. It is generally understood that liposome vesicles are taken up by cells via endocytosis and enter the lysosomal degradation pathway. Thus, some effort towards designing liposomes that avoid degradation has been made. One approach has been to include in the liposome a pH sensitive lipid, such as palmitoylhomocysteine (Connor et al., Proc. Natl. Acad. Sci. (USA) 81:1715 (1984); Chu and Szoka, J. Liposome Res., 4(1):361 (1994)). Such pH sensitive lipids at neutral pH are negatively charged and are stably incorporated into the liposome lipid bilayers. However, at weakly acidic pH (pH less than about 6.8) the lipid becomes neutral in charge and changes in structure sufficiently to destabilize the liposome bilayers. The lipid, when incorporated into a liposome that has been taken into an endosome, where the pH is reported to be between about 5.0 to about 6.0, destabilizes and causes a release of the liposome contents.
The use of cationic lipids, e.g., derivatives of glycolipids with a positively charged ammonium or sulfonium ion-containing headgroup, for delivery of negatively-charged biomolecules, such as oligonucleotides and gene fragments, as a liposome lipid bilayer component is also widely reported. The positively-charged headgroup of the lipid interacts with the negatively-charged cell surface, facilitating contact and delivery of the biomolecule to the cell.
Despite these efforts, delivery of biomolecules, such as oligonucleotides and other materials, as described above, to cells is still lacking in the art. The present invention provides compositions and methods for improving the transfer of an agent, such as a nucleic acid, to cells.